CN114224853A - Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor - Google Patents
Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor Download PDFInfo
- Publication number
- CN114224853A CN114224853A CN202210002403.4A CN202210002403A CN114224853A CN 114224853 A CN114224853 A CN 114224853A CN 202210002403 A CN202210002403 A CN 202210002403A CN 114224853 A CN114224853 A CN 114224853A
- Authority
- CN
- China
- Prior art keywords
- freeze
- stimulating factor
- drying
- stock solution
- recombinant human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/193—Colony stimulating factors [CSF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/12—Carboxylic acids; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
- A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
Abstract
The invention provides a freeze-dried preparation for injection of a PEGylated recombinant human granulocyte stimulating factor, which uses a water-soluble beta-cyclodextrin derivative as a freeze-drying protective agent, and obtains a fluffy dried product by a freeze-drying mode after the split charging of a freeze-dried solution, wherein the prepared product has good stability and high titer, can be stored at room temperature, has low content of aggregated high-molecular protein and low PEG residual quantity, does not contain a surfactant Tween 20 in a prescription, and has higher medication safety; in addition, the preparation process is simple and suitable for large-scale production.
Description
Technical Field
The invention belongs to the technical field of protein and polypeptide medicines, and particularly relates to a freeze-dried preparation for injection of a pegylated recombinant human granulocyte stimulating factor and a preparation method thereof.
Background
Neutropenia and its infectious complications are one of the most common and serious side effects of cytotoxic chemotherapy and other tumor therapies such as radiation therapy, biotherapy and bone marrow transplantation. Cytotoxic chemotherapy works by finding and destroying rapidly growing cells, causing neutropenia due to the high proliferation rate of neutrophil precursors and the rapid metabolic turnover of blood neutrophils. Among chemotherapy patients, the most common symptoms of neutropenia include fever, aphtha and ear infections. Patients with marked neutropenia are often infected with purulent agents, such as sepsis, cellulitis, liver abscess, furuncles, pneumonia, stomatitis, gingivitis, periproctitis, colitis, sinusitis and otitis media. Chemotherapy may be forced to delay until the body produces more neutrophils, which may necessitate lower doses, resulting in less therapeutic effect.
Human granulocyte stimulating factor (G-CSF), a long-chain polypeptide glycoprotein derived from monocytes and fibroblasts, can induce the proliferation and differentiation of hematopoietic stem cells and promote the increase of the number of neutrophils in blood; in addition, the compound has the functions of stimulating the release of mature neutrophils from bone marrow and activating the neutrophils. The human granulocyte stimulating factor (rhG-CSF) produced by recombinant technology is one of the main cell factors for regulating the granulocyte-lineage in the bone marrow, selectively acts on the granulocyte-lineage hematopoietic cells to promote the proliferation and differentiation of the granulocyte-lineage hematopoietic cells, and can increase the functions of the non-differentiated cells in the granulocyte-lineage. Compared with natural products, the biological activity is basically consistent in vivo and in vitro, and the preparation is helpful for preventing neutropenia, reducing the degree of neutropenia, shortening the duration of the agranulocytosis, accelerating the recovery of the number of the agranulocytes, and reducing the risk of fever caused by combined infection. The natural or recombinant G-CSF has a small molecular weight, is easily filtered by glomeruli, has a short circulation half-life in human body of only 2-4 hours, and needs to be injected 1-2 times per day for 5-7 days in each chemotherapy cycle. The polyethylene glycol recombination human granulocyte stimulating factor (PEG-rhG-CSF) is formed by combining the recombination human granulocyte stimulating factor with polyethylene glycol, because the N end of the polyethylene glycol recombination human granulocyte stimulating factor is chemically modified by 20KD monomethoxy polyethylene glycol (mPEG), the chances that the human granulocyte stimulating factor protein is contacted and enzymolyzed by protease in a human body are greatly reduced, compared with the conventional rhG-CSF, the plasma clearance rate is slowed down, the half-life period is obviously prolonged to 46-62h, therefore, the administration times can be reduced in clinical use, the pain of a patient who repeatedly receives injection is avoided, and the polyethylene glycol recombination human granulocyte stimulating factor has the advantages of long acting, less adverse reaction and the like.
Currently, Amgen company in the united states has marketed an injection, which is called Neulasta, and contains polyethylene glycol recombinant human granulocyte stimulating factor, sorbitol, tween 20, acetic acid and sodium acetate, wherein the sorbitol is used for adjusting osmotic pressure, the pH value of acetate buffer salt can be maintained at about 4.0, the tween 20 can prevent main drugs from aggregating, so that the injection is stable, but the tween 20 has stronger hemolytic action and can increase the risk of medication. In addition, the prescription of the currently marketed recombinant human granulocyte stimulating factor freeze-dried powder injection (trade name of glatiramer) is that each recombinant human granulocyte stimulating factor freeze-dried powder injection contains 0.1mg of monododecanoic acid polyoxyethylene sorbitan, 10mg of L-arginine, 10mg of L-phenylalanine, 10mg of L-methionine and 25mg of D-mannitol, the pH value is 6.0-7.5, but the molecular structure of the recombinant human granulocyte stimulating factor is changed after the polyethylene glycol is carried out, in order to protect the recombinant human granulocyte stimulating factor from being inactivated in the freeze-drying process, a new freeze-dried preparation prescription and a new process need to be searched, therefore, Chinese invention patent CN102028661B discloses a polyethylene glycol recombinant human granulocyte stimulating factor freeze-dried powder injection, the technical scheme is mainly that the freeze-dried protective agent is added into the prescription and is prepared by vacuum freeze-drying, the freeze-dried protective agent is selected from one or a plurality of mannitol, arginine and glycine, the obtained product has better stability, but the titer and purity are obviously reduced after long-term or accelerated storage, and the residual quantity of PEG is obviously increased.
Disclosure of Invention
Long-term research and clinical practice show that the PEGylated recombinant human granulocyte colony-stimulating factor injection has the defects of poor stability, short storage period and the like other protein medicaments, and the treatment effect of the PEGylated recombinant human granulocyte colony-stimulating factor injection is influenced. In the storage process, aggregation, PEG shedding and other problems are easy to occur, the safety and the effectiveness of the injection are seriously influenced, and the injection formula contains Tween 20, the Tween 20 has stronger hemolytic effect, can cause potential harm to human bodies after long-term use, and in addition, the storage condition of the finished product is harsh and needs to be stored at 2-8 ℃. According to the invention, a water-soluble beta-cyclodextrin derivative is used as an excipient, and after the freeze-dried stock solution is subpackaged, a fluffy and dry product is obtained in a freeze-drying mode. In addition, the freeze-drying stock solution can be filled into a double-cavity prefilled syringe for freeze-drying, the buffer solution is filled at the other end of the syringe after freeze-drying is finished to serve as a special solvent, the special solvent is injected into the freeze-dried powder during use, medicine waste can be avoided, and the accuracy of the amount of the injected medicine is guaranteed. The freeze-dried preparation for injection prepared by the invention does not contain Tween 20 in the formula, so that the safety of medication is improved, and the obtained finished product can be stored at 25 ℃, so that the storage cost is saved.
Specifically, the invention is realized by the following technical scheme:
a freeze-dried preparation for injection of a pegylated recombinant human granulocyte stimulating factor comprises the pegylated recombinant human granulocyte stimulating factor (PEG-rhG-CSF) and a water-soluble beta-cyclodextrin derivative, wherein the weight ratio of the pegylated recombinant human granulocyte stimulating factor to the water-soluble beta-cyclodextrin derivative is 1: 5 to 25.
Preferably, the dosage ratio of the pegylated recombinant human granulocyte stimulating factor to the water-soluble beta-cyclodextrin derivative in the lyophilized preparation for injection is 1: 15 to 20.
Preferably, the water-soluble beta-cyclodextrin derivative in the freeze-dried preparation for injection is one or a combination of hydroxypropyl beta-cyclodextrin, methyl beta-cyclodextrin, glucose-beta-cyclodextrin and sulfobutyl beta-cyclodextrin; further preferred is hydroxypropyl β cyclodextrin or sulfobutyl β cyclodextrin.
Preferably, the content of the pegylated recombinant human granulocyte stimulating factor in the freeze-dried stock solution of the freeze-dried preparation for injection is 3-20 mg/mL; further preferably, the content of the pegylated recombinant human granulocyte stimulating factor in the freeze-dried stock solution of the freeze-dried preparation for injection is 6-14 mg/mL; in some embodiments, the amount of pegylated human granulocyte stimulating factor in the lyophilized stock solution is 10 mg/mL.
Preferably, the freeze-dried stock solution of the freeze-dried preparation for injection further comprises a buffering agent.
Preferably, the pH value of the freeze-dried preparation for injection is 4.0-5.0.
A preparation method of a freeze-dried preparation for injection of a PEGylated recombinant human granulocyte stimulating factor comprises the following steps: adding the water-soluble beta-cyclodextrin derivative and PEG-rhG-CSF stock solution into a buffer solution, stirring for dissolving, adjusting the pH to 4.0-5.0, diluting to a constant volume with the buffer solution to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, subpackaging, and carrying out vacuum freeze-drying to obtain the beta-cyclodextrin derivative.
Preferably, the buffer is selected from one of citrate buffer or acetate buffer.
Preferably, the concentration of the buffer solution is 10-15 mmol/L. Preferably, the vacuum freeze-drying curve is as follows:
1) pre-freezing: firstly, fully cooling a sample solution to be lyophilized to-40 to-50 ℃ and maintaining for 180 min;
2) sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.15 +/-0.02 mbar, raising the temperature of the sample to-20 ℃ at the speed of 10 ℃/h, and maintaining the temperature for 2-4 hours; later, the temperature is raised to-10 ℃ at the speed of 5 ℃/h, and the temperature is maintained for 10-14 h;
3) and (3) resolving and drying: vacuumizing the freeze dryer and maintaining the vacuum pressure in the freeze dryer at 0.10 +/-0.02 mbar, raising the temperature of the sample to 15-25 ℃ at the speed of 5 ℃/h, continuously drying for 4-6 hours, carrying out ultimate vacuum for 2 hours, carrying out pressure plugging in a vacuum state, and taking out of the box.
Preferably, the freeze-dried stock solution after filtration and sterilization can be filled into a double-cavity prefilled syringe, after freeze-drying is completed, the prepared buffer solution is filled into the other end of the double-cavity prefilled syringe after sterilization and filtration, a plug is added, and a push rod is placed, so that a preparation finished product is obtained.
Preferably, the freeze-drying stock solution after filtration and sterilization can also be filled in a penicillin bottle for freeze-drying, after freeze-drying, the rubber plug is compressed in vacuum, and an aluminum cover is rolled outside to obtain a finished preparation.
The technical scheme adopted by the invention has the following advantages:
(1) the product does not use surfactant Tween 20, and has few adjuvants.
(2) The product has good stability, the obtained preparation product can be stored at room temperature, and the titer and the purity are higher after long-term and accelerated stability investigation.
(3) The product has low content of aggregated high molecular protein, low PEG residue, high safety and good drug effect.
(4) The product is filled in a double-cavity prefilled syringe, and when the product is used, a special solvent is injected into the freeze-dried powder, so that waste is avoided, and the accuracy of the amount of the injected medicine is ensured; the preparation process is simple and suitable for large-scale production.
Detailed Description
The invention is further illustrated by the following examples, which should be properly understood: the examples of the present invention are intended to be illustrative only and not to be limiting, and therefore, the present invention is intended to be simply modified within the scope of the present invention as claimed.
The PEGylated recombinant human granulocyte stimulating factor used in the present invention can be prepared according to patent CN1663962A or other prior art, and other raw materials are commercially available.
Example 1
Prescription:
the preparation method comprises the following steps:
adding a formula amount of sulfobutyl beta cyclodextrin and PEG-rhG-CSF stock solution into a citric acid/sodium citrate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 4.0 by using a hydrochloric acid solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the obtained freeze-dried stock solution into a double-cavity prefilled syringe, and freeze-drying the freeze-dried stock solution in a freeze dryer, wherein the freeze-drying procedure is as follows:
1) pre-freezing: firstly, fully cooling a sample solution to be lyophilized to-40 ℃, and maintaining for 180 min;
2) sublimation drying: vacuumizing in a freeze dryer, maintaining the vacuum pressure at 0.15 +/-0.02 mbar, raising the temperature of a sample to-20 ℃ at 10 ℃/h, and maintaining the temperature for 2 hours; later, the temperature is raised to-10 ℃ at the speed of 5 ℃/h, and the temperature is maintained for 10 h;
3) and (3) resolving and drying: vacuumizing in a freeze dryer and maintaining 0.10 +/-0.02 mbar, raising the temperature of a sample to 15 ℃ at the speed of 5 ℃/h, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, carrying out plug pressing in a vacuum state, and taking out of a box;
and (3) sterilizing and filtering the prepared special solvent for citrate, filling the special solvent for citrate into the other end of the double-cavity prefilled syringe, plugging the special solvent for citrate, and placing a push rod to obtain a finished preparation.
Example 2
Prescription:
the preparation method comprises the following steps:
adding a formula amount of hydroxypropyl beta cyclodextrin and PEG-rhG-CSF stock solution into a citric acid/sodium citrate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 5.0 by using a sodium hydroxide solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain a freeze-dried stock solution, degerming and filtering the obtained freeze-dried stock solution, filling the freeze-dried stock solution into a penicillin bottle, adding a rubber plug into the vial, and freeze-drying the vial, wherein the freeze-drying procedure is as follows:
1) pre-freezing: firstly, fully cooling a sample solution to be lyophilized to-50 ℃, and maintaining for 180 min;
2) sublimation drying: vacuumizing in a freeze dryer, maintaining the vacuum pressure at 0.15 +/-0.02 mbar, raising the temperature of a sample to-20 ℃ at 10 ℃/h, and maintaining the temperature for 4 hours; later, the temperature is raised to-10 ℃ at the speed of 5 ℃/h, and the temperature is maintained for 14 h;
3) and (3) resolving and drying: the freeze dryer was evacuated and maintained at 0.10 + -0.02 mbar, the temperature of the sample was raised to 25 deg.C at 5 deg.C/h, dried for 6 hours, vacuumed for 2 hours again, stoppered under vacuum and removed from the box.
Example 3
Prescription:
the preparation method comprises the following steps:
adding hydroxypropyl beta cyclodextrin and PEG-rhG-CSF stock solution with the formula amount into citric acid/sodium citrate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 4.5 by using sodium hydroxide solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the obtained freeze-dried stock solution into a double-cavity prefilled syringe, and freeze-drying the freeze-dried stock solution in a freeze dryer, wherein the freeze-drying procedure is as follows:
1) pre-freezing: firstly, fully cooling a sample solution to be lyophilized to-45 ℃ and maintaining for 180 min;
2) sublimation drying: vacuumizing in a freeze dryer, maintaining the vacuum pressure at 0.15 +/-0.02 mbar, raising the temperature of a sample to-20 ℃ at 10 ℃/h, and maintaining the temperature for 3 hours; later, the temperature is raised to-10 ℃ at the speed of 5 ℃/h, and the temperature is maintained for 12 h;
3) and (3) resolving and drying: vacuumizing in a freeze dryer and maintaining 0.10 +/-0.02 mbar, raising the temperature of a sample to 20 ℃ at the speed of 5 ℃/h, continuously drying for 4 hours, carrying out ultimate vacuum for 2 hours, carrying out plug pressing in a vacuum state, and taking out of a box;
and (3) sterilizing and filtering the prepared special solvent for citrate, filling the special solvent for citrate into the other end of the double-cavity prefilled syringe, plugging the special solvent for citrate, and placing a push rod to obtain a finished preparation.
Example 4
Prescription:
the preparation method comprises the following steps:
adding a formula amount of sulfobutyl beta cyclodextrin and PEG-rhG-CSF stock solution into a citric acid/sodium citrate buffer solution with the concentration of 10mmol/L, stirring for dissolving, adjusting the pH to 4.0 by using a hydrochloric acid solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the freeze-dried stock solution into a double-cavity prefilled syringe, freeze-drying the freeze-dried stock solution in a freeze dryer, sterilizing and filtering the prepared citrate buffer solution after freeze-drying, filling the sterile filtered citrate buffer solution into the other end of the double-cavity prefilled syringe, plugging, and placing a push rod to obtain a finished preparation, wherein the freeze-drying procedure is the same as that in the embodiment 3.
Example 5
Prescription:
the preparation method comprises the following steps:
adding a formula amount of sulfobutyl beta cyclodextrin and PEG-rhG-CSF stock solution into a citric acid/sodium citrate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 4.5 by using a hydrochloric acid solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the freeze-dried stock solution into a double-cavity prefilled syringe, freeze-drying the freeze-dried stock solution in a freeze dryer, sterilizing and filtering the prepared citrate buffer solution, filling the freeze-dried stock solution into the other end of the double-cavity prefilled syringe, plugging, and placing a push rod to obtain a finished preparation, wherein the freeze-drying procedure is the same as that in the embodiment 3.
Example 6
Prescription:
the preparation method comprises the following steps:
adding a formula amount of glucosyl beta-cyclodextrin and PEG-rhG-CSF stock solution into an acetic acid/sodium acetate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 4.0 by using a hydrochloric acid solution, fixing the volume by using the acetic acid/sodium acetate buffer solution to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the obtained freeze-dried stock solution into a double-cavity prefilled syringe, and freeze-drying the freeze-dried stock solution in a freeze dryer, wherein the freeze-drying procedure is as follows:
1) pre-freezing: firstly, fully cooling a sample solution to be lyophilized to-40 ℃, and maintaining for 5 hours;
2) sublimation drying: vacuumizing in a freeze dryer, maintaining the vacuum pressure at 0.15 +/-0.02 mbar, raising the temperature of a sample to-20 ℃ at 10 ℃/h, and maintaining the temperature for 3 hours; later, the temperature is raised to-15 ℃ at the speed of 5 ℃/h, and the temperature is maintained for 12 h;
3) and (3) resolving and drying: vacuumizing in a freeze dryer and maintaining 0.10 +/-0.02 mbar, raising the temperature of a sample to 20 ℃ at the speed of 5 ℃/h, continuously drying for 8 hours, carrying out ultimate vacuum for 2 hours, carrying out plug pressing in a vacuum state, and taking out of a box;
and sterilizing and filtering the prepared acetate buffer solution, filling the acetate buffer solution into the other end of the double-cavity prefilled syringe, plugging the syringe, and placing a push rod to obtain a finished preparation.
Example 7
Prescription:
the preparation method comprises the following steps:
adding a prescription dose of methyl beta cyclodextrin and PEG-rhG-CSF stock solution into a phosphate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 6.0 by using a sodium hydroxide solution, fixing the volume by using the phosphate buffer solution to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the freeze-dried stock solution into a double-cavity prefilled syringe, putting the syringe into a freeze dryer for freeze-drying, after the freeze-drying is finished, sterilizing and filtering the prepared phosphate buffer solution, filling the sterile filtered phosphate buffer solution into the other end of the double-cavity prefilled syringe, plugging, and placing a push rod to obtain a finished preparation, wherein the freeze-drying procedure is the same as that in the embodiment 3.
Comparative example 1
Prescription:
the preparation method comprises the following steps:
adding hydroxypropyl beta cyclodextrin and PEG-rhG-CSF stock solution with the formula amount into citric acid/sodium citrate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 4.0 by using hydrochloric acid solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, filling the freeze-dried stock solution into a double-cavity prefilled syringe, freeze-drying the freeze-dried stock solution in a freeze dryer, sterilizing and filtering the prepared citrate buffer solution after freeze-drying, filling the sterile filtered citrate buffer solution into the other end of the double-cavity prefilled syringe, plugging, and placing a push rod to obtain a finished preparation, wherein the freeze-drying procedure is the same as that in the embodiment 3.
Comparative example 2
Prescription:
the preparation method comprises the following steps:
adding hydroxypropyl beta cyclodextrin and PEG-rhG-CSF stock solution with the formula amount into citric acid/sodium citrate buffer solution with the concentration of 15mmol/L, stirring for dissolving, adjusting the pH to 4.0 by using hydrochloric acid solution, fixing the volume by using the citric acid/sodium citrate buffer solution to obtain freeze-dried stock solution, degerming and filtering the obtained freeze-dried stock solution, filling the freeze-dried stock solution into a double-cavity prefilled syringe, freeze-drying the freeze-dried stock solution in a freeze dryer, degerming and filtering the prepared citrate buffer solution after freeze-drying, filling the sterile and filtered citrate buffer solution into the other end of the double-cavity prefilled syringe, plugging, and placing a push rod to obtain a finished preparation, wherein the freeze-drying procedure is the same as that in the embodiment 3.
Comparative example 3
1) Preparing a diluent: the used vessels are treated without heat source, the whole process is aseptic, 15g of mannitol, 20g of glycine, 0.492g of glacial acetic acid and 0.148g of sodium acetate are weighed, the volume is accurately determined to 1000mL by using water for injection, the pH value of the diluent is adjusted to 4.0, a 0.22 mu m filter membrane is used for sterilization and filtration, and the endotoxin content of the diluent is determined to be less than 0.25EU/mL by using a limulus reagent method;
2) preparing a semi-finished product: taking a stock solution of the PEGylated recombinant human granulocyte stimulating factor which meets the quality standard, sterilizing and filtering the stock solution by using a 0.22-micron filter membrane, determining the protein concentration, and diluting the stock solution by using a freshly prepared diluent until the concentration of the PEGylated recombinant human granulocyte stimulating factor is 1.0mg/mL to obtain a semi-finished product;
3) filling and freeze-drying: filling the semi-finished product into a washed and sterilized penicillin bottle, adding a rubber plug in the semi-finished product, and putting the penicillin bottle into a freeze dryer for freeze drying, wherein the freeze drying curve is shown in table 1, and the temperature reduction rate in the pre-freezing stage in the vacuum freeze drying process is 2.0 ℃/min;
4) capping, visual inspection and packaging: after the freeze-drying is finished, the rubber plug is pressed under vacuum, the vacuum in the freeze dryer is broken, the sample is taken out, an aluminum cover is rolled outside, unqualified products such as broken bottles and poor rolled covers are removed through visual inspection, and the qualified products are labeled and packaged to obtain finished products.
Table 1 lyophilization profile:
comparative example 4
Solutions were prepared and sterile filtered, with the contents of the various components as shown in table 2, then precision-packaged in 1mL sterile packs and lyophilized in vials. After freeze-drying, the bottle is covered tightly to prepare the PEG-rhG-CSF freeze-dried preparation.
Table 2 recipe:
| PEG-rhG-CSF | phenylalanine | Arginine | Methionine | Mannitol | Tween 20 | Buffer pH |
| 100μg | 10mg | 10mg | 1mg | 25mg | 0.1mg | Phosphate, pH6.5 |
Verification examples
The test samples were subjected to stability examination under the same conditions (25 ℃ RH 60%, 40 ℃ RH 75%, 60 ℃ RH 90%) and the appearance, specific activity, SEC-HPLC, polymer content, reversed phase purity (RP-HPLC) and PEG residue after dissolution were mainly examined.
1. Specific activity
The biological activity of the human granulocyte stimulating factor is measured according to a method for measuring the biological activity of the human granulocyte stimulating factor (NFS-60 cells/MTT colorimetry) (3525 of the four-part general rules of the Chinese pharmacopoeia 2020 edition).
SEC-HPLC and high-molecular protein content
The method is established by HPLC method with reference to the fragment simulation Validation reporting Sep 17,2012, and TSKgel G3000SWXL (7.8mm,30cm,5 μm) is used as a chromatographic column; mobile phase: a solution A-15% ethanol solution (0.272 g of potassium dihydrogen phosphate, 0.532g of disodium hydrogen phosphate and 6.43g of sodium chloride are weighed, a proper amount of water is added for dissolution, the pH value is adjusted to 7.5 by phosphoric acid, water is added for dilution to 1L, a 0.45 mu m filter membrane is used for filtration to obtain a solution A, 850ml of the solution A is taken out, 150ml of chromatographic ethanol is added for uniform mixing to obtain a mobile phase); the flow rate was 1.0ml per minute; column temperature: 25 ℃; cooling type autosampler temperature: 6 ℃, wavelength: 214nm elution mode: gradient eluting for 45 min; sample introduction amount: 100 mu l, calculated by an area normalization method, the area of the main peak of the PEG-rhG-CSF should not be less than 95.0 percent of the total area, and the total amount of the macromolecular protein should not be more than 2.0 percent.
3. Reversed phase purity (RP-HPLC)
The chromatographic column adopts butane silane bonded silica gel as a filler (phenomenex Jupiter C45 μm,250mm multiplied by 4.6mm, or a chromatographic column with equivalent separation efficiency); taking a phase A (trifluoroacetic acid-aqueous solution: measuring 1.0ml of trifluoroacetic acid and adding water to 1000ml, and fully mixing uniformly) and a phase B (trifluoroacetic acid-acetonitrile solution: measuring 1.0ml of trifluoroacetic acid and adding chromatographic pure acetonitrile to 1000ml, and fully mixing uniformly) as mobile phases, carrying out gradient elution for 40 minutes (the phase A is from 60% to 0%, the phase B is from 40% to 100%) at room temperature, wherein the flow rate is 1.5ml per minute, a sample to be tested is diluted into a solution containing 0.5mg in each 1ml by water, the sample amount is 20 mu l, and the detection is carried out at the wavelength of 214 nm. The number of theoretical plates calculated by PEG-rhG-CSF chromatographic peak should not be less than 5000. The area of the main peak of PEG-rhG-CSF is not less than 95.0% of the total area calculated by area normalization method.
PEG residue
The method is established by an HPLC method, and butane silane bonded silica gel is used as a filler (phenomenex Jupiter C45 μm,250mm multiplied by 4.6mm, or a chromatographic column with equivalent separation efficiency); taking phase A (0.1% trifluoroacetic acid-water solution: 1.0ml trifluoroacetic acid is measured and added with water to 1000ml, and fully mixed), phase B (0.1% trifluoroacetic acid-80% acetonitrile solution: 1.0ml trifluoroacetic acid is measured, 800ml chromatographic pure acetonitrile is measured and added with water to 1000ml, and fully mixed) as a mobile phase, wherein the flow rate is 1.0ml per minute, and the column temperature is as follows: room temperature, cooling autosampler temperature: 6 ℃, detector ELSD parameters: (temperature: 113.0 ℃, Impactor: off, carrier gas flow rate 3.1L per minute), sample volume: 100 μ l, gradient elution was performed as in Table 3 below:
table 3:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 80 | 20 |
| 20 | 20 | 80 |
| 35 | 20 | 80 |
| 40 | 80 | 20 |
| 50 | 80 | 20 |
The results of the long-term stability and acceleration stability are shown in tables 4, 5 and 6.
TABLE 4 Long-term stability test results (25 ℃ RH 60%)
TABLE 5 accelerated stability test results (40 ℃ RH 70%)
TABLE 6 accelerated stability test results (60 ℃ RH 90%)
The stability experiment result shows that the titer and purity of the lyophilized preparation for injection of the pegylated recombinant human granulocyte stimulating factor prepared by the invention are obviously higher than those of the prior art after long-term storage for 24 months (25 ℃ 60 RH%), accelerated storage for 3 months (40 ℃ RH 75%) and accelerated storage for 2 weeks (60 ℃ RH 90%), and the content of the high molecular protein and the content of the fallen PEG are obviously lower than those of the prior art, so that the lyophilized preparation has good stability and high safety.
Claims (10)
1. The lyophilized preparation for injection of the pegylated recombinant human granulocyte stimulating factor is characterized by comprising the pegylated recombinant human granulocyte stimulating factor and a water-soluble beta-cyclodextrin derivative, wherein the weight ratio of the pegylated recombinant human granulocyte stimulating factor to the water-soluble cyclodextrin derivative is 1: 5 to 25.
2. The lyophilized formulation for injection according to claim 1, wherein the ratio of the amount of the pegylated recombinant human granulocyte stimulating factor to the amount of the water-soluble β -cyclodextrin derivative is 1: 15 to 20.
3. The lyophilized formulation for injection according to claim 1, wherein the water-soluble β -cyclodextrin derivative is one or more selected from hydroxypropyl β -cyclodextrin, methyl β -cyclodextrin, glucosyl β -cyclodextrin and sulfobutyl β -cyclodextrin.
4. The lyophilized preparation for injection according to claim 1, wherein the content of the pegylated recombinant human granulocyte stimulating factor in the lyophilized stock solution of the lyophilized preparation is 3-20 mg/mL.
5. The lyophilized formulation for injection according to claim 1, further comprising a buffer in a lyophilized stock solution of the lyophilized formulation.
6. The lyophilized preparation for injection according to claim 1, wherein the pH of the lyophilized preparation for injection is 4.0 to 5.0.
7. A method for preparing the lyophilized preparation for injection according to any one of claims 1 to 6, comprising the steps of:
adding the water-soluble beta-cyclodextrin derivative and the stock solution of the pegylated recombinant human granulocyte stimulating factor into a buffer solution, stirring for dissolving, adjusting the pH to 4.0-5.0, using the buffer solution for constant volume to obtain a freeze-dried stock solution, sterilizing and filtering the obtained freeze-dried stock solution, subpackaging, and carrying out vacuum freeze-drying to obtain the compound.
8. The method of claim 7, wherein the buffer is selected from the group consisting of a citrate buffer and an acetate buffer.
9. The method of claim 7, wherein the vacuum freeze-drying profile is as follows:
1) pre-freezing: firstly, fully cooling a sample solution to be lyophilized to-40 to-50 ℃ and maintaining for 180 min;
2) sublimation drying: vacuumizing the freeze dryer and maintaining the vacuum pressure at 0.15 +/-0.02 mbar, raising the temperature of the sample to-20 ℃ at the speed of 10 ℃/h, and maintaining the temperature for 2-4 hours; later, the temperature is raised to-10 ℃ at the speed of 5 ℃/h, and the temperature is maintained for 10-14 h;
3) and (3) resolving and drying: vacuumizing the freeze dryer and maintaining the vacuum pressure in the freeze dryer at 0.10 +/-0.02 mbar, raising the temperature of the sample to 15-25 ℃ at the speed of 5 ℃/h, continuously drying for 4-6 hours, carrying out ultimate vacuum for 2 hours, carrying out pressure plugging in a vacuum state, and taking out of the box.
10. A freeze-dried preparation for injection as claimed in any one of claims 1 to 6, wherein a freeze-dried stock solution containing the pegylated recombinant human granulocyte stimulating factor and the water-soluble beta-cyclodextrin derivative is filled in a double-chamber prefilled syringe after sterilization filtration, and after freeze-drying is completed, the prepared buffer solution is filled in the other end of the double-chamber prefilled syringe after sterilization filtration, and then the other end is plugged and put on a push rod to obtain a finished preparation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210002403.4A CN114224853B (en) | 2022-01-04 | 2022-01-04 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210002403.4A CN114224853B (en) | 2022-01-04 | 2022-01-04 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114224853A true CN114224853A (en) | 2022-03-25 |
| CN114224853B CN114224853B (en) | 2022-09-23 |
Family
ID=80745722
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210002403.4A Active CN114224853B (en) | 2022-01-04 | 2022-01-04 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114224853B (en) |
Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1627958A (en) * | 2002-02-06 | 2005-06-15 | 默克专利股份有限公司 | Immunocytokine-containing lyophilized preparation |
| CN102028661A (en) * | 2010-12-31 | 2011-04-27 | 山东新时代药业有限公司 | Pegylated recombinant human granulocyte colony stimulating factor freeze-dried powder/injection and preparation method thereof |
| CN102670522A (en) * | 2011-03-16 | 2012-09-19 | 江苏泰康生物医药有限公司 | Pharmaceutical preparation containing recombinant human serum albumin-human granulocyte colony stimulating factor fusion protein and preparation thereof |
| CN104689333A (en) * | 2007-08-27 | 2015-06-10 | 拉蒂奥法姆有限责任公司 | Liquid formulation of G-CSF conjugate |
| CN104906053A (en) * | 2015-06-12 | 2015-09-16 | 北京四环生物制药有限公司 | Human recombinant granulocyte colony-stimulating factor freeze-dried powder injection for injection |
| CN105273076A (en) * | 2014-06-18 | 2016-01-27 | 江苏奥赛康药业股份有限公司 | Preparation method and pharmaceutical composition of PEGylated recombinant human granulocyte colony-stimulating factor |
| CN106986928A (en) * | 2016-04-15 | 2017-07-28 | 江苏恒瑞医药股份有限公司 | A kind of purification process of PEG-rhG-CSF |
| CN107129531A (en) * | 2016-04-15 | 2017-09-05 | 江苏恒瑞医药股份有限公司 | A kind of purification process of PEG-rhG-CSF |
| CN107188952A (en) * | 2016-05-17 | 2017-09-22 | 江苏恒瑞医药股份有限公司 | A kind of purification process of recombinant human granulocyte colony stimulating factor |
| CN108368162A (en) * | 2016-04-15 | 2018-08-03 | 江苏恒瑞医药股份有限公司 | A kind of renaturation and purification process of recombined human granulocyte stimulating factors |
| CN112121009A (en) * | 2020-09-24 | 2020-12-25 | 科兴生物制药股份有限公司 | New preparation of polyethylene glycol modified recombinant human granulocyte stimulating factor |
| CN112394115A (en) * | 2019-08-19 | 2021-02-23 | 鲁南制药集团股份有限公司 | Method for detecting decoration degree of polyethylene glycol (PEG) repairman of PEGylated recombinant granulocyte stimulating factor |
| CN113368063A (en) * | 2020-03-10 | 2021-09-10 | 鲁南制药集团股份有限公司 | Recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein freeze-dried preparation for injection and preparation method thereof |
| CN113797171A (en) * | 2021-04-28 | 2021-12-17 | 山东新时代药业有限公司 | Pegylated recombinant human granulocyte colony stimulating factor freeze-dried preparation |
-
2022
- 2022-01-04 CN CN202210002403.4A patent/CN114224853B/en active Active
Patent Citations (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1627958A (en) * | 2002-02-06 | 2005-06-15 | 默克专利股份有限公司 | Immunocytokine-containing lyophilized preparation |
| CN104689333A (en) * | 2007-08-27 | 2015-06-10 | 拉蒂奥法姆有限责任公司 | Liquid formulation of G-CSF conjugate |
| CN102028661A (en) * | 2010-12-31 | 2011-04-27 | 山东新时代药业有限公司 | Pegylated recombinant human granulocyte colony stimulating factor freeze-dried powder/injection and preparation method thereof |
| CN102670522A (en) * | 2011-03-16 | 2012-09-19 | 江苏泰康生物医药有限公司 | Pharmaceutical preparation containing recombinant human serum albumin-human granulocyte colony stimulating factor fusion protein and preparation thereof |
| CN105273076A (en) * | 2014-06-18 | 2016-01-27 | 江苏奥赛康药业股份有限公司 | Preparation method and pharmaceutical composition of PEGylated recombinant human granulocyte colony-stimulating factor |
| CN104906053A (en) * | 2015-06-12 | 2015-09-16 | 北京四环生物制药有限公司 | Human recombinant granulocyte colony-stimulating factor freeze-dried powder injection for injection |
| CN106986928A (en) * | 2016-04-15 | 2017-07-28 | 江苏恒瑞医药股份有限公司 | A kind of purification process of PEG-rhG-CSF |
| CN107129531A (en) * | 2016-04-15 | 2017-09-05 | 江苏恒瑞医药股份有限公司 | A kind of purification process of PEG-rhG-CSF |
| CN108368162A (en) * | 2016-04-15 | 2018-08-03 | 江苏恒瑞医药股份有限公司 | A kind of renaturation and purification process of recombined human granulocyte stimulating factors |
| CN107188952A (en) * | 2016-05-17 | 2017-09-22 | 江苏恒瑞医药股份有限公司 | A kind of purification process of recombinant human granulocyte colony stimulating factor |
| CN112394115A (en) * | 2019-08-19 | 2021-02-23 | 鲁南制药集团股份有限公司 | Method for detecting decoration degree of polyethylene glycol (PEG) repairman of PEGylated recombinant granulocyte stimulating factor |
| CN113368063A (en) * | 2020-03-10 | 2021-09-10 | 鲁南制药集团股份有限公司 | Recombinant leukocyte inhibitory factor and hirulog peptide chimeric protein freeze-dried preparation for injection and preparation method thereof |
| CN112121009A (en) * | 2020-09-24 | 2020-12-25 | 科兴生物制药股份有限公司 | New preparation of polyethylene glycol modified recombinant human granulocyte stimulating factor |
| CN113797171A (en) * | 2021-04-28 | 2021-12-17 | 山东新时代药业有限公司 | Pegylated recombinant human granulocyte colony stimulating factor freeze-dried preparation |
Non-Patent Citations (1)
| Title |
|---|
| 张培彪: "聚乙二醇化重组人粒细胞刺激因子工作对照品的制备和标定", 《食品与药品》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114224853B (en) | 2022-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4496537A (en) | Biologically stable alpha-interferon formulations | |
| TWI606838B (en) | Daptomycin compositions and related methods | |
| EA013324B1 (en) | Bendamustine pharmaceutical compositions for lyophilization | |
| EP2974716B1 (en) | Spray-dried powder comprising vancomycin hydrochloride, and industrial manufacturing method thereof | |
| EP3218061B1 (en) | Carmustine pharmaceutical composition | |
| CN101578106A (en) | HGF preparation | |
| US20210353719A1 (en) | Microparticle Formulations for Delivery to the Lower and Central Respiratory Tract and Methods of Manufacture | |
| ES2905874T3 (en) | Polyalkylene Oxide Asparaginase Formulations and Methods of Making and Using The Same | |
| CN108379561A (en) | A kind of PEGylated uricoxidase freeze dried powder and preparation method thereof | |
| AU2013336206A1 (en) | Stable pharmaceutical composition of peginterferon alpha-2b | |
| JP3714951B2 (en) | IL-6-containing pharmaceutical composition | |
| CN114224853B (en) | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor | |
| CN107028897B (en) | Immune regulator thymopentin powder injection medicine composition and quality control method | |
| EP3423042B1 (en) | A lyophilised pharmaceutical formulation and its use | |
| CN113797171B (en) | Pegylated recombinant human granulocyte colony stimulating factor freeze-dried preparation | |
| KR880002037B1 (en) | Interferon composition and its preparation method | |
| CN102028661B (en) | Pegylated recombinant human granulocyte colony stimulating factor freeze-dried powder/injection and preparation method thereof | |
| CN117651548A (en) | Freeze-dried preparation solution, freeze-dried preparation, method and application | |
| CN113368063B (en) | Recombinant leukocyte inhibitory factor and hirudin chimeric protein freeze-dried preparation for injection and preparation method thereof | |
| EP3054924B1 (en) | Stable pharmaceutical formulations of caspofungin | |
| CN111465389B (en) | Pharmaceutical composition of docetaxel conjugate and preparation method thereof | |
| RU2404797C1 (en) | COMPOSITION CONTAINING HUMAN RECOMBINANT INTERFERON ALPHA-2b, AND MEDICINAL AGENT OF PROLONGED ACTION FOR VETERINARY SCIENCE ON ITS BASIS, HAVING ANTIVIRUS AND IMMUNOMODULATORY ACTION | |
| CN111450242A (en) | Bivalirudin injection preparation and preparation method and application thereof | |
| WO2022116134A1 (en) | Fosphenytoin sodium solid composition, lyophilization method, and use of fosphenytoin sodium solid composition | |
| CN103877010A (en) | Preparation method of Romidepsin solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |